1. Field of the Invention
The present invention relates to a fluorescence detection apparatus for observing the fluorescence from a sample dyed with two or more different fluorescent dyes on a fluorescent tagged sample.
2. Description of the Related Art
In the conventional life science field, the observation of fluorescence from a fluorescent tagged sample has been widely performed. In the fluorescence observation, incident-light illumination is commonly used. In the incident-light illumination, a excitation light does not directly enter the optical path for detection of fluorescence, but the excitation light reflected or scattered by a sample or an optical system is mixed into the optical path for detection. Generally, since an excitation light is much stronger than fluorescence, it is difficult to detect the fluorescence when the slightest excitation light is mixed. Therefore, it is necessary to completely separate the fluorescence from the excitation light.
A method of detecting fluorescence, a method of obtaining fluorescence in a desired wavelength range by cutting off unnecessary light such as an excitation light, etc. using an optical filter at a stage preceding a photodetector is used. In this case, the optical filter separates an excitation light from fluorescence based on the difference in wavelength.
Recently, since fluorescent dyes_and fluorescent protein having various fluorescent spectra_have been sequentially developed and improved, not only fluorescence in a specific wavelength range is obtained using an optical filter as in a conventional method, but also fluorescence can be spectrally detected using a dispersion element such as a grating (diffraction grating), etc., and the wavelength characteristic data (spectrum) of fluorescence can be obtained.
For example, Japanese Published Patent Application No. Hei 8-43739 discloses a method of detecting a desired wavelength by dispersing fluorescence from a sample by a grating and leading it to a detector.
Additionally, U.S. 2002/21440 discloses the configuration of dispersing fluorescence from a sample by a prism and leading it to a detector. By changing the incident angle of the fluorescence entering a prism and continuously changing the range of a wavelength entering the detector, the spectral distribution of the fluorescence can be measured.
According to US2002/21440, a fluorescent spectrum can be continuously measured. However, the case in which a sample dyed with multiple dyes is pumped by an excitation light having two or more wavelengths, and the fluorescence is to be spectrally observed is not referred to in the documents of the prior art.